PXD050935

PXD050935 is an original dataset announced via ProteomeXchange.

Title	Keap1 is an endogenous dCas9 suppressor inhibiting CRISPRa and CRISPRi efficiency
Description	As a potent and accurate genome-editing tool, CRISPR-Cas9 has been widely used in biomedical research and evaluated as gene therapy in treating human diseases. Although distinct engineered Cas9s, dCas9s and additional endonucleases have been identified, as these bacterial enzymes do not naturally express in mammalian cells, whether and how bacterial Cas9 proteins are regulated by mammalian hosts remains poorly understood. Here, we identified Keap1 as an endogenous E3 ligase that targets Cas9/dCas9/Fanzor1 for ubiquitination and degradation. Cas9-“ETGE” mutants evading Keap1 recognition displayed enhanced gene editing ability in cells. dCas9-“ETGE” mutants displayed extended protein half-life on chromatin, leading to significantly improved CRISPa and CRISPRi efficacy. Cas9 binding to Keap1 also inactivate Keap1 function via competing with Keap1 substrates or binding partners, while engineered Cas9 mutants showed less perturbation. Thus, our study reveals a mammalian specific Cas9 regulation and provides new Cas9 designs not only with enhanced gene regulatory capacity but also with minimal effects on disrupting endogenous Keap1 signaling.
HostingRepository	PRIDE
AnnounceDate	2025-01-20
AnnouncementXML	Submission_2025-01-20_09:26:00.293.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Dennis Goldfarb
SpeciesList	scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationList	No PTMs are included in the dataset
Instrument	Orbitrap Fusion Lumos

Revision	Datetime	Status	ChangeLog Entry
0	2024-03-24 13:55:20	ID requested
⏵ 1	2025-01-20 09:26:00	announced

10.1093/NAR/GKAE761;

submitter keyword: CRISPR, KEAP1, CAS9

Pengda Liu
contact affiliation	Lineberger Comprehensive Cancer Center, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA. Department of Biochemistry and Biophysics, School of Medicine, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
contact email	pengda_liu@med.unc.edu
lab head
Dennis Goldfarb
contact affiliation	Cell Biology and Physiology Institute for Informatics
contact email	d.goldfarb@wustl.edu
dataset submitter

Dataset FTP location NOTE: Most web browsers have now discontinued native support for FTP access within the browser window. But you can usually install another FTP app (we recommend FileZilla) and configure your browser to launch the external application when you click on this FTP link. Or otherwise, launch an app that supports FTP (like FileZilla) and use this address: ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2025/01/PXD050935

PRIDE project URI

• PRIDE
  1. PXD050935
     1. Label: PRIDE project
     2. Name: Keap1 is an endogenous dCas9 suppressor inhibiting CRISPRa and CRISPRi efficiency